Developing dual and specific inhibitors of dimethylarginine dimethylaminohydrolase-1 and nitric oxide synthase: toward a targeted polypharmacology to control nitric oxide.

Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712, USA.

Abstract

Molecules that block nitric oxide's (NO) biosynthesis are of significant interest. For example, nitric oxide synthase (NOS) inhibitors have been suggested as antitumor therapeutics, as have inhibitors of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catabolizes endogenous NOS inhibitors. Dual-targeted inhibitors hold promise as more effective reagents to block NO biosynthesis than single-targeted compounds. In this study, a small set of known NOS inhibitors are surveyed as inhibitors of recombinant human DDAH-1. From these, an alkylamidine scaffold is selected for homologation. Stepwise lengthening of one substituent converts an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor and then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N(5)-(1-iminohexyl)-l-ornithine for neuronal NOS (1.7, 3, 20, >1,900 microM, respectively) and DDAH-1 (990, 52, 7.5, 110 microM, respectively). A 1.9 A X-ray crystal structure of the N5-(1-iminopropyl)-L-ornithine:DDAH-1 complex indicates covalent bond formation between the inhibitor's amidino carbon and the active-site Cys274, and solution studies show reversible competitive inhibition, consistent with a reversible covalent mode of DDAH inhibition by alkylamidine inhibitors. These represent a versatile scaffold for the development of a targeted polypharmacological approach to control NO biosynthesis.

The active site of DDAH-1 in complex with L-IPO (13) and omit map density. Atoms colored in cyan, blue, red and yellow for carbon, nitrogen, oxygen and sulfur, respectively. Active-site residues and L-IPO are labeled. The Fo-Fc electron density map, with L-IPO and the C274 Sγ atom not included in the calculated structure factors, is shown at 3 σ in blue.

Comparison of DDAH-1 and NOS active sites. The left panel shows a superimposition of human DDAH-1 with bound L-IPO (13) (green) and L-257 (16) (pink). The right panel shows a superimposition of bovine eNOS bound by L-NIO (12) (green) and rat nNOS bound by Nω-propyl-l-arginine (pink), a compound of comparable length to N5-(1-iminopentyl)-l-ornithine (14). Inhibitors are shown in ball and stick format and colored by heteroatom as described earlier. The heme groups of NOS are shown in stick models. Surface features (light blue) are shown for the L-257-DDAH-1 complex and the Nω-propyl-l-arginine-nNOS complex to highlight the active-site cavity shape. Figures are constructed using coordinates from this work and protein data bank accession codes 2JAJ, 1ED6 and 1MMV (, , ).